Sister centromere fusion is a process unique to meiosis that promotes co-orientation of the sister kinetochores, ensuring they attach to microtubules from the same pole during metaphase I. We have found that the kinetochore protein SPC105R/KNL1 and Protein Phosphatase 1 (PP1-87B) regulate sister centromere fusion in Drosophila oocytes. The analysis of these two proteins, however, has shown that two independent mechanisms maintain sister centromere fusion. Maintenance of sister centromere fusion by SPC105R depends on Separase, suggesting cohesin proteins must be maintained at the core centromeres. In contrast, maintenance of sister centromere fusion by PP1-87B does not depend on either Separase or WAPL. Instead, PP1-87B maintains sister centromeres fusion by regulating microtubule dynamics. We demonstrate that this regulation is through antagonizing Polo kinase and BubR1, two proteins known to promote stability of kinetochore-microtubule (KT-MT) attachments, suggesting that PP1-87B maintains sister centromere fusion by inhibiting stable KT-MT attachments. Surprisingly, C(3)G, the transverse element of the synaptonemal complex (SC), is also required for centromere separation in Pp1-87B RNAi oocytes. This is evidence for a functional role of centromeric SC in the meiotic divisions, that might involve regulating microtubule dynamics. Together, we propose two mechanisms maintain co-orientation in Drosophila oocytes: one involves SPC105R to protect cohesins at sister centromeres and another involves PP1-87B to regulate spindle forces at end-on attachments.

The chromosomes in the oocytes of many animals appear to promote bipolar spindle assembly. In Drosophila oocytes, spindle assembly requires the chromosome passenger complex (CPC), which consists of INCENP, Borealin, Survivin, and Aurora B. To determine what recruits the CPC to the chromosomes and its role in spindle assembly, we developed a strategy to manipulate the function and localization of INCENP, which is critical for recruiting the Aurora B kinase. We found that an interaction between Borealin and the chromatin is crucial for the recruitment of the CPC to the chromosomes and is sufficient to build kinetochores and recruit spindle microtubules. HP1 colocalizes with the CPC on the chromosomes and together they move to the spindle microtubules. We propose that the Borealin interaction with HP1 promotes the movement of the CPC from the chromosomes to the microtubules. In addition, within the central spindle, rather than at the centromeres, the CPC and HP1 are required for homologous chromosome bi-orientation.

There is an interest in identifying Anaphase Promoting-Complex/Cyclosome (APC/C) inhibitors that lead to sensitivity to microtubule poisons as a strategy for targeting cancer cells. Using budding yeast Saccharomyces cerevisiae, peptides derived from the Mitotic Arrest Deficient 2 (Mad2)-binding motif of Cell Division Cycle 20 (Cdc20) were observed to inhibit both Cdc20- and CDC20 Homology 1 (Cdh1)-dependent APC/C activity. Over expression of peptides in vivo led to sensitivity to a microtubule poison and, in a recovery from a microtubule poison arrest, delayed degradation of yeast Securin protein Precocious Dissociation of Sisters 1 (Pds1). Peptides with mutations in the Cdc20 activating KILR-motif still bound APC/C, but lost the ability to inhibit APC/C in vitro and lost the ability to induce sensitivity to a microtubule poison in vivo. Thus, an APC/C binding and activation motif that promotes mitotic progression, namely the Cdc20 KILR-motif, can also function as an APC/C inhibitor when present in excess. Another activator for mitotic progression after recovery from microtubule poison is p31comet, where a yeast predicted open-reading frame YBR296C-A encoding a 39 amino acid predicted protein was identified by homology to p31comet, and named Tiny Yeast Comet 1 (TYC1). Tyc1 over expression resulted in sensitivity to microtubule poison. Tyc1 inhibited both APC/CCdc20 and APC/CCdh1 activities in vitro and bound to APC/C. A homologous peptide derived from human p31comet bound to and inhibited yeast APC/C demonstrating evolutionary retention of these biochemical activities. Cdc20 Mad2-binding motif peptides and Tyc1 disrupted the ability of the co-factors Cdc20 and Cdh1 to bind to APC/C, and co-over expression of both together in vivo resulted in an increased sensitivity to microtubule poison. We hypothesize that Cdc20 Mad2-binding motif peptides, Tyc1 and human hp31 peptide can serve as novel molecular tools for investigating APC/C inhibition that leads to sensitivity to microtubule poison in vivo.

Cryptococcus neoformans is a ubiquitously distributed human pathogen. It is also a model system for studying fungal virulence, physiology and differentiation. Light is known to inhibit sexual development via the evolutionarily conserved white collar proteins in C. neoformans. To dissect molecular mechanisms regulating this process, we have identified the SSN8 gene whose mutation suppresses the light-dependent CWC1 overexpression phenotype. Characterization of sex-related phenotypes revealed that Ssn8 functions as a negative regulator in both heterothallic a-α mating and same-sex mating processes. In addition, Ssn8 is involved in the suppression of other physiological processes including invasive growth, and production of capsule and melanin. Interestingly, Ssn8 is also required for the maintenance of cell wall integrity and virulence. Our gene expression studies confirmed that deletion of SSN8 results in de-repression of genes involved in sexual development and melanization. Epistatic and yeast two hybrid studies suggest that C. neoformans Ssn8 plays critical roles downstream of the Cpk1 MAPK cascade and Ste12 and possibly resides at one of the major branches downstream of the Cwc complex in the light-mediated sexual development pathway. Taken together, our studies demonstrate that the conserved Mediator protein Ssn8 functions as a global regulator which negatively regulates diverse physiological and developmental processes and is required for virulence in C. neoformans.

The chromosomes in the oocytes of many animals appear to promote bipolar spindle assembly. In Drosophila oocytes, spindle assembly requires the chromosomal passenger complex (CPC), which consists of INCENP, Borealin, Survivin and Aurora B. To determine what recruits the CPC to the chromosomes and its role in spindle assembly, we developed a strategy to manipulate the function and localization of INCENP, which is critical for recruiting the Aurora B kinase. We found that an interaction between Borealin and HP1 is crucial for the initial recruitment of the CPC to the chromosomes and is sufficient to build kinetochores and recruit spindle microtubules. We also found that HP1 moves from the chromosomes to the spindle microtubules along with the CPC, and based on this, propose a mechanism for how the CPC moves from the chromosomes to the microtubules. Within the central spindle, rather than at the centromeres, the CPC and HP1 are required for homologous chromosome bi-orientation.